Developing molecular tools for the genetic manipulation of Nannochloropsis
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The main aim of the master project was the establishment of the efficient protocols for the genetic manipulation (nuclear transformation and chloroplast transformation) of Nannochloropsis oceanica CCMP1779. Nannochloropsis is a heterokont algae and a potential producer of biofuel and high value lipids, as it can accumulate significant amount of triacylglycerols. Cell concentration is an essential parameter for establishing an electroporation protocol. Therefore, a convenient method for the determination of cell concentration was established. Determination of cell concentrations by hemocytometer, flow cytometer and optical density at 750 nm (OD750nm) were compared with one another. Satisfactory correlation between flow cytometer and OD750nm as an indicator of cell growth was established by preparing the accurate standard curves. It was revealed that the maximum cell density that could be reliably determined by the flow cytometer was 5.85×106 cells/mL corresponding to an OD750nm value of 0.1169. It was evaluated under what conditions cells could be harvested and stored and counted at a later point. A storage protocols that used glutaraldehyde and snap-freeze in liquid nitrogen decreased the number of counted cells by less than 4 and 3% respectively, compared to the number of cells counted in fresh samples.To achieve nuclear transformation of N. oceanica CCMP1779, the plasmid pSELECT100 was used. As this plasmid contains a hygromycin resistance cassette, the hygromycin concentration that prevented growth of untransformed wild type cells on agar plates was assessed. These experiments revealed that hygromycin concentration of 30-50 µg/mL resulted in no growth. Consequently, the selective medium for the screening of transformants contained 50 µg/mL hygromycin. Parameters that result in efficient nuclear transformation, such as harvesting cell concentrations, cell washing steps, voltage and resistance applied under electroporation, incubation of the transformed cells, and addition of the carrier DNA, were investigated. The experiments revealed that growing the cells up to an exponential phase concentration of 2-4×106 cells/mL resulted in competent for transformation cells. The highest transformation efficiency achieved at the experiment was 4.08×10-5 colonies/cells/µg, which was a higher efficiency compared to the available transformation protocols. A voltage of 1800 volts and resistance of 500 Ohm and a relative low amount of the transformation plasmid DNA (~1 µg) resulted in the highest transformation efficiencies. Genetic manipulation of the chloroplast genome by biolistic transformation (particle bombardment) has been established in several microalgae. As no published protocols exist for the chloroplast transformation of Nannochloropsis, the initial steps to develop a protocol, which are based on integrating an antibiotic resistance cassette into the chloroplast genome, were made.A wide range of antibiotics was evaluated to establish which antibiotic inhibits cell growth at a low concentration, in addition to availability of antibiotic resistance cassette in the lab at the Department of Biotechnology. Growth experiments on agar plates supplemented with different antibiotics (ampicillin, apramycin, chloramphenicol, cycloheximide, gentamycin, G418 neomycin, hygromycin, kanamycin, spectinomycin, and tetracaine) at different concentrations revealed that chloramphenicol inhibits growth at ≥ 20 µg/mL. Consequently, agar-solidified selective medium containing chloramphenicol at a concentration of 40 µg/mL was used to select for cells transformed by particle bombardment.Modern DNA sequence assembly (Gibson Assembly) was carried out to generate a plasmid (pLit_chlL_chlor) that carried the chloramphenicol resistance gene flanked by sequences of the chlL gene that is native to N. oceanica CCMP1779. The choice of the gene was based on the assessment that heterokont algae also encode a gene within the nucleus catalyzing the same reaction (i.e. conversion of protochlorophyllide to chlorophyllide). This makes the deletion of the chlL gene unimportant for the cell growth under constant light supply.The transformation of the chloroplast genome with the plasmid pLit_chlL_chlor was performed with biolistic delivery technology using tungsten microparticles coated with the plasmid DNA. The technique was previously reported being more efficient for plastid transformation by homologous recombination. The screening of the potential transformants on the selective medium resulted in appearance of few colonies indicating the successful transformation. However, the incubation of the cell colonies in the selective liquid medium did not result in any cell growth, leaving the confirmation of the successful insertion of the chloramphenicol resistance cassette as an experiment of future research.